SCCA is an antigen extracted from squamous epithelial cells that demonstrates high concentrations in blood obtained from patients suffering from squamous cell carcinoma of the cervix, lungs, esophagus and skin and is frequently used to diagnosis squamous cell carcinoma (H. Kato et al., Cancer, 40: 1621-1628 (1977); N. Mino et al., Cancer, 62: 730-734 (1988)). Since SCCA levels in the blood demonstrate a favorable correlation with such factors as the progressive stage of squamous cell carcinoma, degree of malignancy and tumor size in particular, it is a particularly effective marker not only for early detection of cancer but also for evaluating the effects of cancer treatment and diagnosing the risk of recurrence.
In addition, increased expression of SCCA is also known to be observed in the upper layer of psoriatic epidermis (Takeda A. et al., J. Invest. Dermatol. (2002) 118(1), 147-154). Psoriasis is a type of skin disease in the form of chronic and recurrent inflammatory parakeratosis characterized by abnormal proliferation and differentiation of epidermal cells and infiltration by inflammatory cells. Psoriasis is believed to occur due to genetic factors in addition to various environmental factors (Hopso-Havu et al., British Journal of Dermatology (1983) 109, 77-85).
SCCA is encoded by two genes SCCA-1 and SCCA-2 arranged in tandem on chromosome 18q21.3. The proteins SCCA-1 and SCCA-2 encoded by these genes both have a molecular weight of about 45,000, and although they have an extremely high degree of homology, since they have different amino acid sequences at the reaction site, they are believed to have different functions (Schick et al., J. Biol. Chem. (1997) 27231, 1849-55). Although SCCA-1 and SCCA-2 are known to be highly expressed in diseases such as squamous cell carcinoma and psoriasis, it is unclear as to what functions they perform in diseased cells.
On the other hand, keratinocytes are known to have the function of forming a protective barrier referred to as the “cornified layer” against harmful environments as a result of terminal differentiation. The terminal differentiation process is accurately controlled by a differentiation program, and begins with proliferative basal cells, goes through the stages of prickle cells, granular cells and finally ends with the differentiation into keratinocytes. Dramatic changes occur both inside and outside the keratinocytes during the transition period from granular cells to keratinocytes. The keratinocytes lose their nucleus and cellular organelles, while acquiring a peripheral lipid layer, a strengthened cell membrane referred to as cornified integument, and a keratin pattern. The keratin pattern maintains a flexible and tight internal structure. According to previous reports, keratinocytes undergoing differentiation demonstrate characteristics of apoptosis such as DNA fragmentation and TUNEL-positive cells (Haake A. R., J. Invest. Dermatol., 101, 107-12 (1993)). Caspase-like activity has been detected in extracts of human keratinocytes, and several types of caspases are expressed in human keratinocytes. However, other reports have indicated that typical pro-apoptotic caspases such as caspase-3, caspase-6 and caspase-7 are not activated at the time of terminal differentiation. Differentiation abnormalities frequently lead to the permanent presence of nuclei in the cornified layer referred to as “parakeratosis”. Parakeratosis causes serious damage to the barrier function of the skin. However, it has yet to be determined as to which factors are involved in the denucleation process, and the manner in which this process is regulated during keratinocyte differentiation.
Caspases are well-known apoptotic cell death execution factors, and are cysteine proteases preserved in the evolutionary process that cleave substrates after aspartic acid residues. Caspases in mammals are divided into three subgroups according to their structure and function, namely initiator caspase, effector caspase and inflammatory caspase. Effector caspase fulfills the important role of decomposing the inhibitor ICAD of CAD (caspase activated DNase), resulting in the dissociation of CAD as an active nuclease (Enari M. et al., Nature, 391, 43-50 (1998)). Caspase activity is regulated by various molecules. In particular, there are three groups of inhibitory proteins in direct collaboration with several caspases. Baculovirus anti-apoptotic protein p35 inhibits caspases-1, -3, -6, -7, -8 and -10 without acting on serine or other cysteine proteinases (Zhou Q. et al., Biochemistry, 37, 10757-65 (1998)). Baculovirus also synthesizes other anti-apoptotic proteins and inhibitors of apoptosis proteins (IAP). Homologues of IAP are also found in mammals (Verhagen A. M. et al., Genome Biol., 2, Reviews 3009 (2001)). Mammalian IAP blocks apoptosis by inhibiting caspase-14 or by antagonizing pro-apoptosis-promoting factors such as DIABLO/Smac (Wu G., Nature, 408, 1008-12 (2000)). Cytokine response modifier A (Crm A) is a genetic product of cowpox virus that is capable of inhibiting apoptotic caspases and inflammatory caspases (Garcia-Calvo M. et al., J. Biol. Chem., 273, 32608-13 (1998)). It is quite interesting that Crm A has been suggested to belong to a superfamily of serine proteinase inhibitors, and that some serine proteinase inhibitors, such as PI-9 and PAI-1, are able to inhibit apoptosis by interacting with caspase-1 and caspase-3, respectively (Annand R. R. et al., Biochem. J., 342Pt3, 655-65 (1999)). It will therefore be interesting to determine whether or not terminal differentiation of keratinocytes constitutes a portion of apoptosis phenomena, and the manner in which arbitrary regulatory proteins are involved in this process.
Caspase-14 is the newest member of the caspase family, and is expressed nearly exclusively by differentiating keratinocytes. Caspase-14 was identified by an EST homologous among members of the caspase family. According to findings of recent research, the caspase-14 present in keratinocytes is a processed heterodimer that demonstrates enzymatic activity with respect to the synthetic substrate Trp-Glu-His-Asp-AFC corresponding to caspase-1 (Mikolajczyk J. et al., Biochemistry, 43, 10560-9 (2004)). This hydrolysis activity requires protein decomposition and cleavage as well as the presence of a kosmotropic salt. Although the primary structure of caspase-14 is extremely similar to that of inflammatory caspases such as caspase-1, -4 and -5, the expression of caspase-14 limited to differentiated keratinocytes has been suggested to be involved in keratinocyte terminal differentiation in a different mode (Lippens S. et al., Cell Death Differ., 7, 1218-24 (2001)). However, the activation mechanism of caspase-14 along with its natural substrate or regulatory factors have yet to be elucidated.